A number of infectious diseases and cancers lacks efficient therapies. Monoclonal antibodies have generally not been successful against these targets, partly due to variability of the complex targets and adaptive mutations of target proteins causing immune escape from monoclonal antibody recognition. Polyclonal antibodies on the other hand are able to target a plurality of dynamic targets, e.g., on viruses or cancer cells. Also, polyclonal antibodies have the highest probability of retaining activity in the event of antigenic mutation.
Different commercially available polyclonal antibody therapeutics exist including: 1) normal human immunoglobulin isolated from the blood of normal human donors; 2) human hyperimmune immunoglobulin derived from the blood of individual human donors carrying antibodies against a particular disease target, e.g., a virus, which they previously have encountered either through infection or vaccination; and 3) animal hyperimmune immunoglobulin derived from the blood of immunized animals.
Immunoglobulin purified from human blood has proved effective against infections with hepatitis B virus, respiratory syncytial virus, cytomegalovirus and other herpes viruses, rabies virus, botulinum toxin, etc, as well as in the neonatal rhesus D prophylaxis. Immunoglobulin purified from the blood of rabbits immunized with human T cells is used to afford T cell immunosuppression in the treatment or prevention of transplant rejection (e.g., Thymoglobulin). Normal human immunoglobulin has been utilized to boost the immune system of immunodeficient patients, as well as in the therapy of various autoimmune disorders.
Nevertheless, widespread immunoglobulin use has been limited due to the constrained supply of donor blood raw material, problems with batch-to-batch variations, and variable safety. Animal-derived immunoglobulins in particular are faced with the same problems of immunogenicity as was observed for animal-derived monoclonal antibodies in the 1980s and 1990s. Finally, as with other blood products, the risk of transmission of infectious agents such as HIV, herpes or hepatitis viruses or prions remains. Accordingly, while clinicians acknowledge that polyclonal antibodies are a preferred therapeutic in some situations, their use has been very limited.
New approaches to generate human immunoglobulins arose with the transgenic animal techniques. Transgenic mice carrying human immunoglobulin loci have been created (U.S. Pat. No. 6,111,166). These mice produce fully human immunoglobulins, and antibodies against a specific target can be raised by usual immunization techniques. However, larger antibody yields are limited because of the relatively small size of mice. Larger animals have also been made transgenic for the human immunoglobulin genes, e.g., cows, sheep, rabbits, and chickens (Kurolwa, Y. et al. Nature Biotechnology; 2002; 20: 889-893). However, producing polyclonal antibodies for therapy from the blood of such animals is not without complications. First, the immunophysiology of the animal and humans may display considerable differences, causing a difference in the resulting immune repertoire, functional rearrangement, and diversity of the antibody response. Second, mitotic instability of the introduced immunoglobulin loci might influence the long-term production of antibodies. Third, it is technically challenging to delete the animal's own immunoglobulin loci so that e.g., the animal antibody production will not exceed the production of human antibody. Fourth, the risk of transmission of infectious agents such as viruses, prions or other pathogens accompanies the administration of human antibodies produced in animals.
Accordingly, there is a need for manufacturing technologies for producing recombinant polyclonal proteins, such as antibodies, in sufficiently large amounts and with minimal batch-to-batch variations for safe clinical uses. Efficient methods for manufacturing homogenous recombinant proteins using eukaryotic (in particular mammalian) expression cell lines have been developed for the production of a variety of proteins including monoclonal antibodies, interleukins, interferons, tumor necrosis factor, coagulation factors VII, VIII and IX. Many of these techniques are based on transfection and random integration of the gene of interest into the genome of the expression cell line followed by selection, amplification, and characterization of a high-producer expression clone and propagation of this clone as a master expression cell line.
The expression of an inserted foreign gene may be influenced by “position effects” from surrounding genomic DNA. In many cases, the gene is inserted into sites where the position effects are strong enough to inhibit the synthesis of the product of the introduced gene. Furthermore, the expression is often unstable due to silencing mechanisms (i.e. methylation) imposed by the surrounding chromosomal host DNA.
Systems allowing integration and expression of a gene of interest in mammalian cells at a specific genomic location have been developed for the expression of a homogenous recombinant protein composition (U.S. Pat. Nos. 4,959,317 and 5,654,182; WO 98/41645; WO 01/07572). WO 98/41645 describes the site-specific integration for production of a mammalian cell line that secretes, for example, antibody. However, this expression is monoclonal and there is no indication that transfections could be done with a library of vectors. Nor are there any suggestions how to maintain the original diversity generated by specific VH-VL combinations in a library.